The gram-positive spore-forming obligate-pathogen Bacillus anthracis (also referred to as B. anthracis) represents the etiological agent of anthrax, a currently rare disease in humans, yet potentially associated with intentional bioterror use. In the most severe respiratory form, B. anthracis infection is initiated by inhalation of spores which germinate into fast dividing vegetative cells that secrete toxins and virulence factors during growth in the host, resulting in massive bacteremia and consequently generalized systemic failure and death.
The lethality of anthrax has been attributed to two main aspects of B. anthracis pathogenesis: the activity of the bacterial exotoxins and the remarkable proliferous nature of the bacteria in the host. B. anthracis secretes two exotoxins, Lethal Toxin (LT) and Edema Toxin (ET) composed of binary combinations of the three proteins Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). PA, the common subunit of both toxins, is not harmful by itself, yet it plays the essential role of binding to a receptor on the surface of host target cells and mediating the intracellular translocation of the lethal subunits of the toxin complex—LF (a zinc protease which together with PA forms the exotoxin LT) and EF (an adenylate cyclase which together with PA constitutes the exotoxin ET). PA elicits a protective immune response and therefore its administration represents the basis for all preventive anthrax countermeasures (Chitlaru et al., 2011) both for pre and post-exposure prophylaxis. The three components of the toxin, are encoded by genes located on pXO1, one of the two virulence plasmids naturally harbored by B. anthracis. A second well-established virulence factor is represented by a poly D-glutamate anti-phagocytic capsule synthesized by enzymes encoded by genes located on the second native plasmid pXO2. The virulence of B. anthracis is governed by regulatory factors which affect the synthesis of the virulence determinants and which coordinate cross-talk circuits linking chromosome and plasmid located genes.
Anthrax is acknowledged as a toxinogenic disease, owing to the lethality of its major toxins. Yet, during infection, B. anthracis secretes a large number of proteins, many of which bear biological functions indicative of a role in the onset and progression of the disease.
For all living organisms, quality control of protein synthesis is a vital activity. One central player in the context of protein quality control is represented by the HtrA (High Temperature Requirement A) family of serine proteases, which are structurally and functionally conserved across a wide range of evolutionary distinct phylogenetic classes both in prokaryots and eukaryots. HtrA proteins exhibit the dual biological activities of chaperones and proteases, and as such, bacterial HtrA has a known function in the turnover of damaged or malfolded proteins that may deleteriously accumulate, particularly under stress conditions. The HtrA family of proteins (often referred as Deg or Do serine protease) was originally identified in E. coli as essential for the response to heat shock, and subsequently shown to consist of the three chaperons DegP, DegQ and DegS, active in the correct folding and/or in the proteolytic processing and maturation of some proteins, as well as in the degradation of abnormal exported polypeptides. HtrA proteins, exhibit a characteristic structure, composed of an N-terminal trypsin-like serine protease domain and at least one C-terminal PDZ domain which recognizes substrates and activates the protease function. In E. coli and B. subtillis, the HtrA family of proteases are important for the survival of the bacteria under different stress regimens. In addition, in Gram positive bacteria, the HtrA chaperones/proteases are closely associated with the SecA membrane-translocation machinery. For example, in Streptococcus piogenes, HtrA is localized at the ExportA membranal complex dedicated to the biogenesis of secreted proteins. In some cases, HtrA was invoked as being directly involved in the proteolytic processing or secretion of specific virulence-associated proteins such as SpeB and Hemolysin in Streptococcus pyogenes, Pertussis toxin S1 and possibly Adhesin P1 of Streptococcus mutans. Furthermore, attenuated Salmonella enterica mutated in the htrA gene are effective live vaccines as well as possible vectors for delivery of recombinant heterologous antigens, compatible with human use.
The search for bacterial proteins fulfilling functions which sustain the outstanding ability of B. anthracis to expand in the host represented the objective of extensive genomic-proteomic-serologic screens of B. anthracis surface and secreted proteomes (reviewed in Chitlaru et al., 2009, and in Shaferman et al., 2010). Based on these studies, a list of immunogenic proteins could be assembled, many of which representing potential novel B. anthracis virulence factors (Chitlaru et al., 2007; Gat et al. 2006) for subsequent studies addressing their ability to serve as the basis for future improved vaccines, therapeutic intervention and diagnostics. HtrA was distinguished in these studies (i) by its assignment to a regulon which comprises secreted proteins exhibiting a pattern of expression similar to that of the toxin (elevated expression in the presence of CO2), (ii) by its high immunogenicity which indicates in-vivo expression in the host, and (iii) by the observation that significant amounts of HtrA can be detected in the circulation of infected animals relatively early in infection (WO2010/109451; Sela Abramovich et al., 2009). By examining the phenotype associated with disruption of the htrA gene, the present invention provides evidence that HtrA is directly or indirectly involved in the export of some bacterial secreted factors (other than the bacterial toxin), is required for the resistance of B. anthracis to stress conditions, it is unexpectedly essential for manifestation of B. anthracis virulence, and most importantly B. anthracis strains in which htrA gene was disrupted represent efficacious and safe immunization means for providing protection against B. anthracis infection.
It is an object of the present invention to provide an isolated Bacillus anthracis (B. anthracis) strain in which the htrA gene of said B. anthracis is silenced. Thus, encompassed by the invention is such a strain in which the htrA gene is mutated by the removal of said gene.
It is another object of the invention to provide an isolated Bacillus anthracis (B. anthracis) strain in which the htrA gene of said B. anthracis is silenced, which can be used for the preparation of a vaccine.
It is yet another object of the invention to provide an isolated B. anthracis strain in which the htrA gene of said B. anthracis is disrupted, which can be used as a platform for expression and protective immunization with homologous and heterologous bacterial antigens